CN111307976B - High-throughput detection method for carotenoid in aquatic product - Google Patents
High-throughput detection method for carotenoid in aquatic product Download PDFInfo
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Abstract
The invention discloses a high-flux detection method of carotenoid in aquatic products, which comprises the following steps: A. pre-treating; B. detecting a sample solution to be detected by a high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrometer to obtain a high performance liquid chromatogram of the sample solution to be detected; and C, analyzing the data. The high-flux detection method for the carotenoids in the aquatic products disclosed by the invention has the following beneficial effects: 1. the determination lower limit of the carotenoid is 0.05mg/kg, and the standard curve is good in the linear range of 10 ng/mL-200 ng/mL; 2. the pretreatment combines solid phase extraction and matrix dispersion purification technologies, and the extraction and purification work of 3 carotenoids with greatly different physicochemical properties is realized through one-time pretreatment, so that the detection efficiency is improved, and the expensive time and the reagent cost consumption are reduced; 3. the method is applied to aquatic products, and 50 carotenoid species are detected in total.
Description
Technical Field
The invention relates to the field of detection of nutrient components of aquatic products, in particular to a high-throughput detection method of carotenoids in aquatic products.
Background
Carotenoids are natural pigment molecules, mainly exist in aquatic organisms such as fish, shrimps, crabs and shells, and the structural composition and distribution of the carotenoids are important indexes for evaluating the quality of the aquatic products. The photosensitive carotenoid can be used as a coloring agent for organisms and tissues to improve the sensory quality, and can also enhance the adaptability of aquatic organisms to the environment and some important physiological functions, such as seedling raising efficiency, disease resistance and the like. The research shows that: the aquatic organisms cannot synthesize the carotenoid from the head, but can convert or directly absorb the dietary carotenoid, each organism has the carotenoid which is unique, and even has the specificity among different tissues, which is one of the reasons for more kinds of the carotenoid in the aquatic products.
Carotenoids are of a wide variety and are divided into three major classes according to their chemical structures: the first type is free carotene, which has no oxygen in the molecule, is highly unsaturated hydrocarbon, and is orange, such as alpha, beta-carotene; the second group is the oxygenated carotenes, which are oxygenated derivatives of carotenes containing one or more oxygen molecules in the ring at both ends of the molecule, the most common modifying groups being hydroxyl, methoxy, carbonyl, carboxyl, etc., such as lutein, astaxanthin, etc., the first two groups being collectively referred to as non-esterified carotenoids; the third class is esters of carotenes, including mono-and di-esters, which are the result of re-esterification of the free carotenoid with endogenous fatty acids.
The traditional pretreatment technology for detecting carotenoid in aquatic products is mainly direct solvent extraction. The technology only adopts a single solvent for extraction (acetone is mostly adopted), has the defect of selectivity, and particularly can not be compatible with small molecular free carotenoid and metabolites thereof and low-polarity carotenoid ester at the same time; and the method does not carry out purification treatment aiming at the attribute of the extracting solution matrix, is only suitable for an analysis method for detecting a certain type of carotene in the fixed food matrix, and has low sample treatment flux. The carotenoid is of various types, and the carotenoid and metabolites thereof in a plurality of aquatic products are not separated and identified, so that a comprehensive and efficient pretreatment method is established, which is an important premise for ensuring that unknown compounds are identified, and has very important significance for perfecting the carotenoid types in the aquatic products and exploring the metabolic pathways thereof. Therefore, a more comprehensive and efficient method for detecting carotenoid species in aquatic products is needed.
A commonly used instrumental analysis method for carotenoids, comprising: liquid phase-ultraviolet visible light absorption spectrum and liquid phase-triple quadrupole mass spectrum. Most carotenoids have three maximum absorption peaks in ultraviolet light, commonly called "three-finger peaks", and the total amount of carotenoids can be analyzed, but the existence form (free, esterified and oxidized) of carotenoids in a matrix cannot be further identified. At present, the carotenoid research mostly adopts a liquid phase-triple quadrupole mass spectrometry technology, which needs characteristic ion fragments of a known compound to be detected, and data is acquired in a multi-reaction ion detection mode in a targeted manner, so that qualitative and quantitative analysis of the known compound is realized.
Disclosure of Invention
The invention aims to overcome the defects of selectivity defects, few detection types, low detection efficiency and the like of the existing method for detecting carotenoid in aquatic products, and provides a high-throughput detection method suitable for carotenoid in aquatic products.
Considering that the coverage of carotenoid is of various types, the polarity difference between different types is large; and the aquatic product has high-protein and high-fat matrix properties, so that a strong matrix effect is generated, and the detection result is seriously deviated. Therefore, according to the pKa value and the polarity range of the extraction solvent, the method adopts fractional purification extraction: the first stage adopts methanol oscillation extraction and HLB column purification, and can realize effective extraction and separation of non-esterified carotenoid (most of free carotenoid and oxygen-containing carotenoid); the second stage adopts methanol extraction, dichloromethane auxiliary extraction and neutral alumina powder purification, and can realize the extraction and separation of the esterified carotenoid while removing grease. The high-throughput detection of the common carotenoids in the aquatic products can be realized by combining the first and second-stage extracting solutions and performing data analysis by adopting high performance liquid chromatography-quadrupole electrostatic field orbital trap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS).
The technical scheme is as follows: a high-throughput detection method for carotenoids in aquatic products comprises the following steps:
A. pretreatment:
(A1) weighing 1g of aquatic product to be detected, adding the aquatic product to be detected into 10mL of methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, performing ultrasonic extraction for 20min, and centrifuging at 4-10 ℃ for 5-10 min at a rotating speed of 12000r/min to obtain an organic phase and a residue;
(A2) taking the organic phase obtained in the step (A1), adding at least 4 times of volume of water into the organic phase to obtain a sample loading solution, purifying the sample loading solution by an HLB (hydrophile-lipophile balance) column to obtain a purified sample loading solution, concentrating the purified sample loading solution to less than 2.0ml by nitrogen blowing, and carrying out volume metering to 2.0ml by a methanol solution containing 0.1 wt% of HF to obtain a purified sample loading solution with a constant volume, wherein:
the HLB column purification uses methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol as eluent to elute;
(A3) adding the residue obtained in the step (A1) into 10mL of methanol-dichloromethane solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, performing ultrasonic extraction for 20min, centrifuging for 5-10 min at 4-10 ℃ at the rotating speed of 12000r/min, and obtaining an organic phase and residue, wherein:
In the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1: 1;
(A4) taking the organic phase obtained in the step (A3), blowing nitrogen to concentrate until the volume is not changed, then fixing the volume to 2.0ml through a methanol solution containing 0.1 wt% of HF, adding 1g of neutral alumina powder, fully shaking, centrifuging at 4-10 ℃ for 5-10 min at a rotating speed of 12000r/min, and obtaining a supernatant and a precipitate after completion;
(A5) mixing the purified sample solution with the constant volume obtained in the step (A2) and the supernatant obtained in the step (A4), and then filtering the mixture through a filter membrane with the pore diameter of 0.22 mu m to obtain a sample solution to be detected;
B. and (C) detecting the sample solution to be detected obtained in the step (A5) by using a high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrometer to obtain a high performance liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows:
high performance liquid phase parameters:
column ZORBAX Eclipse XDB C8 column 2.1mm x 100mm, particle size 3.5 μm;
mobile phase A: an aqueous solution containing 0.1% formic acid +2mM ammonium formate;
mobile phase B: a methanol solution containing 0.1 v/v% formic acid +15 v/v% isopropanol was injected at a flow rate of 0.8mL/min, a sample size of 5.0. mu.L, a split ratio of 1:4, and the gradient was as follows:
positive and negative ion mode gradient elution procedure
Mass spectrum parameters: the mass spectrum is subjected to full-scan measurement in a positive/negative ion conversion mode, and the mass range is as follows: m/z 200-1500, resolution 70000, and target value of automatic gain control 5 × e5(ii) a Anion mode 2900V, positive ion mode 3800V, ion transfer tube temperature 350 ℃, desolvation gas nitrogen flow rate: 35L/h, flow rate of auxiliary gas nitrogen: 15L/h, the temperature of a gasification chamber is 400 ℃; respectively carrying out positive and negative ion correction on the instrument before the sample runs; the second stage adopts an automatic trigger mode, the resolution is 35,000, and the target value of automatic gain control is 2 × e5Range of collision energy10-80%, and the retention time is +/-1.0 min according to the retention time of a target;
c data analysis
C1 plots standard curves for the standards:
preparing mixed standard working solutions with different concentrations by taking alpha-carotene, beta-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combretastatin, cryptoxanthin, diadinoxanthin, dihydrolutein, echinenone, fucoxanthin alcohol, neoxanthin, polymethacrylic acid alcohol, uric acid lactone, mussel flavin, zeaxanthin, scallop alcoholic ketone, diatoxanthine, hydroxyl echinenone, dinotefuran, pectic xanthin, cryptoxanthin, astaxanthin linoleate monoester and astaxanthin dipalmitate, testing according to the same conditions of the step B to obtain a high-efficiency liquid chromatogram of the standard working solution, and drawing a standard curve according to the concentration and peak area of each standard product to obtain a standard curve of the standard product;
C2 selecting standard curve of linoleic acid astaxanthin monoester or dipalmitoyl astaxanthin ester with similar structure according to the quantity of fatty acid chains for relative quantitative analysis of carotenoid mono-and diester without standard;
c3, calculating the concentration of each component to be detected in the sample solution to be detected according to the high performance liquid chromatogram of the sample solution to be detected obtained in the step (B), the corresponding standard curve of the standard product, the linoleic acid astaxanthin monoester standard curve and the dipalmitate astaxanthin ester standard curve, and calculating the content of the carotenoid in the aquatic product to be detected according to a formula (1):
X＝C*V/m*1000
in the formula:
the content of the substance to be detected in the X-sample is mg/kg;
the concentration of the substance to be measured in the C-sample treatment solution is calculated according to a standard curve, a linoleic acid astaxanthin monoester standard curve or a dipalmitate astaxanthin ester standard curve, and the unit is mug/L
V is volume of constant volume, and the unit is mL;
m-sample volume or mass in g.
Further, the aquatic product to be detected in the step (a1) is one of fish, shrimp, crab, and shell.
Further, the concentrations of the different concentrations of the mixed standard working solution in step C1 were 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 200ng/mL, respectively.
Further, the carotenoid esters without standards in step C2 include astaxanthin diester-20: 3/20:1, astaxanthin diester-20: 3/18:2, astaxanthin diester-22: 6/18:1, astaxanthin diester-20: 4/20:5, astaxanthin diester-20: 5/20:5, astaxanthin diester-22: 6/16:0, astaxanthin diester-22: 6/16:1, astaxanthin diester-20: 5/16:0, astaxanthin diester-22: 6/14:0, astaxanthin diester-20: 5/16:1, astaxanthin diester-18: 1/16:0, astaxanthin diester-16: 0/16:0, astaxanthin diester-22: 6/20:5, astaxanthin diester-20: 5/16:1, Astaxanthin diester-20: 5/18:1, astaxanthin monoester-18: 2, astaxanthin monoester-16: 0, astaxanthin monoester-18: 1, astaxanthin monoester-22: 6, astaxanthin monoester-20: 1, astaxanthin monoester-20: 5, astaxanthin monoester-16: 1, astaxanthin monoester-20: 4, astaxanthin diester-22: 6/16:0, adonixanthin diester-22: 6-20:6, and adonixanthin diester-22: 6-16: 0.
The high performance liquid chromatography-quadrupole electrostatic field Orbitrap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS) technology adopted by the application is different from the conventional triple quadrupole mass spectrometry in that the high performance liquid chromatography-quadrupole electrostatic field Orbitrap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS) technology does not need to know the characteristic ion fragment of a compound to be detected, but directly adopts high precision mass number (resolution ratio is 70000, m/z 200) to carry out directional and non-directional full scanning detection on a sample to be detected, does not need repeated sample introduction when a target compound needs to be added, and is capable of re-analyzing the existing full scanning data, thereby being particularly suitable for multi-component simultaneous screening and qualitative and quantitative analysis of unknown substances.
Has the advantages that: the high-flux detection method for the carotenoids in the aquatic products disclosed by the invention has the following beneficial effects:
1. the determination lower limit of the carotenoid is 0.05mg/kg, and the standard curve is good in the linear range of 10 ng/mL-200 ng/mL (the regression coefficient R2 is more than 0.99);
2. the sample pretreatment combines the solid phase extraction and matrix dispersion purification technology, and the extraction and purification work of common 3 carotenoids with greatly different physical and chemical properties is realized through one-time pretreatment, so that the detection efficiency is greatly improved, and the expensive time and the reagent cost consumption are reduced;
3. the method is applied to aquatic products, 50 carotenoid species are detected in total, and 25 non-esterified carotenoids are detected: free carotenoids 2 and oxygen-containing carotenoids 23; and 25 esterified carotenoids: 14 astaxanthin diester, 8 astaxanthin monoester, 1 astaxanthin diester and 2 calendula flavin diester can be simultaneously and accurately detected at one time, the detected carotenoid types and content are superior to those of the existing detection method, and the method has very important significance for perfecting the carotenoid types in aquatic products and exploring the metabolic pathways of the carotenoid types.
Drawings
FIG. 1 is a flow chart of the high throughput detection method of carotenoids in aquatic products disclosed by the present invention.
The specific implementation mode is as follows:
the following is a detailed description of specific embodiments of the invention.
Instruments and reagents (Instruments and materials)
Q-active quadrupole electrostatic field orbitrap high resolution mass spectrometer (Semmer Feishel science ThermoFisher Scientific Co.) was equipped with a source of H-ESI II. The liquid chromatography system is an UltiMate3000 high pressure liquid chromatography band autosampler. Column ZORBAX Eclipse XDB C8 column (2.1mm X100 mm, particle size 3.5 um). Milli-Q high purity water generators (Millipore, USA). A freeze dryer (SIGMA, Germany). Refrigerated centrifuge (SIGMA, germany). Vortex vibrators (Heldolph, Germany). Filter (DIKMA, PTFE 0.22 μm). Waters Oasis HLB column (6cc/200mg, methanol activation before loading, water balance).
Ammonium formate and formic acid (chromatographically pure, Sigma-Aldrich, USA). Other reagents (chromatographically pure, Merck, Germany). The experimental water was Milli-Q ultrapure water (18.2. omega. M). The carotenoid standards were purchased from Sigma and Dr. Ehrenstontorfer and were more than 95% pure. The method comprises the following steps: alpha-carotene, beta-carotene, astaxanthin, fucoxanthin, zeaxanthin, canthaxanthin, lutein, combretastatin, cryptoxanthin, diadinoxanthin, dihydrolutein, echinenone, fucoxanthin alcohol, neoxanthin, polymethacrylin, polymethacrylxanthin alcohol, uric acid lactone, musselxanthin, zeaxanthin, scallop alcohol ketone, diatoxanthin, hydroxyechinenone, diadinoxanthin, pectinxanthin, cryptoxanthin; esterified carotenoids include: linoleic acid astaxanthin monoester, dipalmitoyl astaxanthin ester.
Standard stock solutions: weighing appropriate amount of standard substances, dissolving astaxanthin dipalmitate with dichloromethane, adding methanol to desired volume, dissolving the rest with methanol to obtain 0.1mg/mL standard stock solution, and storing at-20 deg.C in dark.
Standard working solution: and respectively sucking 100 mu L of the standard stock solution of each component, putting the standard stock solution into a 10mL volumetric flask, and respectively metering the volume to the scale with methanol, wherein the concentration is 1.0 mu g/mL.
Detailed description of the preferred embodiment 1
A high-flux detection method for carotenoids in aquatic products comprises the following steps:
A. pretreatment:
(A1) weighing 1g of aquatic product to be detected, adding the aquatic product to be detected into 10mL of methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, ultrasonically extracting for 20min, and centrifuging at 4 ℃ for 10min at the rotating speed of 12000r/min to obtain an organic phase and residue;
(A2) taking the organic phase obtained in the step (A1), adding at least 4 times of volume of water into the organic phase to obtain a sample loading solution, purifying the sample loading solution by an HLB (hydrophile-lipophile balance) column to obtain a purified sample loading solution, concentrating the purified sample loading solution to less than 2.0ml by nitrogen blowing, and carrying out volume metering to 2.0ml by a methanol solution containing 0.1 wt% of HF to obtain a purified sample loading solution with a constant volume, wherein:
the HLB column purification adopts methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol as eluent for elution;
(A3) Adding the residue obtained in the step (A1) into 10mL of methanol-dichloromethane solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, performing ultrasonic extraction for 20min, centrifuging at 4 ℃ for 10min at the rotating speed of 12000r/min, and obtaining an organic phase and residue, wherein:
in the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1: 1;
(A4) taking the organic phase obtained in the step (A3), blowing nitrogen to concentrate until the volume is not changed, then fixing the volume to 2.0ml through a methanol solution containing 0.1 wt% of HF, adding 1g of neutral alumina powder into the organic phase, fully shaking the mixture, centrifuging the mixture at 10 ℃ for 10min at the rotating speed of 12000r/min, and obtaining supernatant and precipitate after the centrifugation is finished;
(A5) combining the purified sample solution obtained in the step (A2) after constant volume and the supernatant obtained in the step (A4), and then passing through a filter membrane with the aperture of 0.22 mu m to obtain a sample solution to be detected;
B. and (C) detecting the sample solution to be detected obtained in the step (A5) by using a high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrometer to obtain a high performance liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows:
high performance liquid phase parameters:
column ZORBAX Eclipse XDB C8 column 2.1mm X100 mm, particle size 3.5 μm;
A mobile phase A: an aqueous solution containing 0.1% formic acid +2mM ammonium formate;
mobile phase B: a methanol solution containing 0.1 v/v% formic acid +15 v/v% isopropanol,
the flow rate is 0.8mL/min, the sample injection amount is 5.0 mu L, the split ratio is 1:4, and the gradient is shown in the following table:
positive and negative ion mode gradient elution procedure
Mass spectrum parameters: the mass spectrum is subjected to full-scan measurement in a positive/negative ion conversion mode, and the mass range is as follows: m/z 200-1500, resolution 70000, and target value of automatic gain control 5 × e5(ii) a Negative ion mode 2900V, positive ionSubmode 3800V, ion transfer tube temperature 350 ℃, desolvation gas nitrogen flow rate: 35L/h, flow rate of auxiliary gas nitrogen: 15L/h, the temperature of a gasification chamber is 400 ℃; respectively carrying out positive and negative ion correction on the instrument before the sample runs; the second stage adopts an automatic trigger mode, the resolution is 35,000, and the target value of automatic gain control is 2 × e5The collision energy range is 10-80%, and the retention time is +/-1.0 min according to the retention time of the target;
c data analysis
C1 plots standard curves for the standards:
preparing mixed standard working solutions with different concentrations by taking alpha-carotene, beta-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combretastatin, cryptoxanthin, diadinoxanthin, dihydrolutein, echinenone, fucoxanthin alcohol, neoxanthin, polymethacrylic acid alcohol, uric acid lactone, mussel flavin, zeaxanthin, scallop alcoholic ketone, diatoxanthine, hydroxyl echinenone, dinotefuran, pectic xanthin, cryptoxanthin, astaxanthin linoleate monoester and astaxanthin dipalmitate, testing according to the same conditions of the step B to obtain a high-efficiency liquid chromatogram of the standard working solution, and drawing a standard curve according to the concentration and peak area of each standard product to obtain a standard curve of the standard product;
C2 selecting standard curve of linoleic acid astaxanthin monoester or dipalmitoyl astaxanthin ester with similar structure according to the quantity of fatty acid chains for relative quantitative analysis of carotenoid mono-and diester without standard;
c3, calculating the concentration of each component to be detected in the sample solution to be detected according to the high performance liquid chromatogram of the sample solution to be detected obtained in the step (B), the corresponding standard curve of the standard product, the linoleic acid astaxanthin monoester standard curve and the dipalmitate astaxanthin ester standard curve, and calculating the content of the carotenoid in the aquatic product to be detected according to a formula (1):
X＝C*V/m*1000
in the formula:
the content of the substance to be detected in the X-sample is mg/kg;
the concentration of the substance to be measured in the C-sample treatment solution is calculated according to a standard curve, a linoleic acid astaxanthin monoester standard curve or a dipalmitate astaxanthin ester standard curve, and the unit is mug/L
V is volume of constant volume, and the unit is mL;
m-sample volume or mass in g.
Further, the aquatic product to be detected in the step (a1) is fish.
Further, the concentrations of the different concentrations of the mixed standard working solution in step C1 were 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 200ng/mL, respectively.
Further, the carotenoid esters without standards in step C2 include astaxanthin diester-20: 3/20:1, astaxanthin diester-20: 3/18:2, astaxanthin diester-22: 6/18:1, astaxanthin diester-20: 4/20:5, astaxanthin diester-20: 5/20:5, astaxanthin diester-22: 6/16:0, astaxanthin diester-22: 6/16:1, astaxanthin diester-20: 5/16:0, astaxanthin diester-22: 6/14:0, astaxanthin diester-20: 5/16:1, astaxanthin diester-18: 1/16:0, astaxanthin diester-16: 0/16:0, astaxanthin diester-22: 6/20:5, Astaxanthin diester-20: 5/18:1, astaxanthin monoester-18: 2, astaxanthin monoester-16: 0, astaxanthin monoester-18: 1, astaxanthin monoester-22: 6, astaxanthin monoester-20: 1, astaxanthin monoester-20: 5, astaxanthin monoester-16: 1, astaxanthin monoester-20: 4, astaxanthin diester-22: 6/16:0, adonixanthin diester-22: 6-20:6, and adonixanthin diester-22: 6-16: 0.
Specific example 2
A high-flux detection method for carotenoids in aquatic products comprises the following steps:
A. pretreatment:
(A1) weighing 1g of aquatic product to be detected, adding the aquatic product to be detected into 10mL of methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, ultrasonically extracting for 20min, and centrifuging at 10 ℃ for 5min at the rotating speed of 12000r/min to obtain an organic phase and residue;
(A2) Taking the organic phase obtained in the step (A1), adding at least 4 times of volume of water into the organic phase to obtain a sample loading solution, purifying the sample loading solution by an HLB (hydrophile-lipophile balance) column to obtain a purified sample loading solution, concentrating the purified sample loading solution to less than 2.0ml by nitrogen blowing, and carrying out volume metering to 2.0ml by a methanol solution containing 0.1 wt% of HF to obtain a purified sample loading solution with a constant volume, wherein:
the HLB column purification uses methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol as eluent to elute;
(A3) adding the residue obtained in the step (A1) into 10mL of methanol-dichloromethane solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, performing ultrasonic extraction for 20min, centrifuging for 5min at 10 ℃ at the rotating speed of 12000r/min, and obtaining an organic phase and residue, wherein:
in the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1: 1;
(A4) taking the organic phase obtained in the step (A3), blowing nitrogen to concentrate until the volume is not changed, then fixing the volume to 2.0ml through a methanol solution containing 0.1 wt% of HF, adding 1g of neutral alumina powder into the organic phase, fully shaking, centrifuging at 10 ℃ for 5min at the rotating speed of 12000r/min, and obtaining supernatant and precipitate after completion;
(A5) combining the purified sample solution obtained in the step (A2) after constant volume and the supernatant obtained in the step (A4), and then passing through a filter membrane with the aperture of 0.22 mu m to obtain a sample solution to be detected;
B. Detecting the sample solution to be detected obtained in the step (A5) by using a high performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometer to obtain a high performance liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows:
high performance liquid phase parameters:
column ZORBAX Eclipse XDB C8 column 2.1mm X100 mm, particle size 3.5 μm;
a mobile phase A: an aqueous solution containing 0.1% formic acid +2mM ammonium formate;
and (3) mobile phase B: a methanol solution containing 0.1 v/v% formic acid +15 v/v% isopropanol,
the flow rate is 0.8mL/min, the sample injection amount is 5.0 mu L, the split ratio is 1:4, and the gradient is shown in the following table:
positive and negative ion mode gradient elution procedure
Mass spectrum parameters: the mass spectrum is subjected to full-scan measurement in a positive/negative ion conversion mode, and the mass range is as follows: m/z 200-1500, resolution 70000, and target value of automatic gain control 5 × e5(ii) a Anion mode 2900V, positive ion mode 3800V, ion transfer tube temperature 350 ℃, desolvation gas nitrogen flow rate: 35L/h, flow rate of auxiliary gas nitrogen: 15L/h, the temperature of a gasification chamber is 400 ℃; respectively carrying out positive and negative ion correction on the instrument before the sample runs; the second stage adopts an automatic trigger mode, the resolution is 35,000, and the target value of automatic gain control is 2 × e5The collision energy range is 10-80%, and the retention time is +/-1.0 min according to the retention time of the target;
C data analysis
C1 plots standard curves for the standards:
preparing mixed standard working solutions with different concentrations by taking alpha-carotene, beta-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combretaxanthin, cryptoxanthin, diadinoxanthin, dihydrolutein, echinenone, fucoxanthin alcohol, neoxanthin, polymethacrylic acid, polymethacrylflavin alcohol, uric acid lactone, mussel flavin, zeaxanthin, scallop alcohol ketone, diatoxanthin, hydroxyl echinenone, dinoflagellate flavin, pectic xanthin, cryptoxanthin, linoleic acid astaxanthin monoester and dipalmitate astaxanthin ester, testing according to the same conditions in the step B to obtain high-efficiency liquid chromatogram of the standard working solutions, and drawing standard curves for each standard product according to the concentration and peak area to obtain the standard curves of the standard products;
c2 selecting standard curve of linoleic acid astaxanthin monoester or dipalmitoyl astaxanthin ester with similar structure according to the quantity of fatty acid chains for relative quantitative analysis of carotenoid mono-and diester without standard;
c3, calculating the concentration of each component to be detected in the sample solution to be detected according to the high performance liquid chromatogram of the sample solution to be detected obtained in the step (B), the corresponding standard curve of the standard product, the linoleic acid astaxanthin monoester standard curve and the dipalmitate astaxanthin ester standard curve, and calculating the content of the carotenoid in the aquatic product to be detected according to a formula (1):
X＝C*V/m*1000
In the formula:
the content of the substance to be detected in the X-sample is mg/kg;
c-the concentration of the substance to be detected in the sample treatment solution is calculated according to a standard curve, a linoleic acid astaxanthin monoester standard curve or a dipalmitate astaxanthin ester standard curve, and the unit is mu g/L
V is volume of constant volume, unit is mL;
m-sample volume or mass in g.
Further, the aquatic product to be detected in the step (a1) is a crab. Of course, in another embodiment, the aquatic product to be detected in step (a1) is a shell.
Further, the concentrations of the different concentrations of the mixed standard working solution in step C1 were 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 200ng/mL, respectively.
Further, the carotenoid esters without standards in step C2 include astaxanthin diester-20: 3/20:1, astaxanthin diester-20: 3/18:2, astaxanthin diester-22: 6/18:1, astaxanthin diester-20: 4/20:5, astaxanthin diester-20: 5/20:5, astaxanthin diester-22: 6/16:0, astaxanthin diester-22: 6/16:1, astaxanthin diester-20: 5/16:0, astaxanthin diester-22: 6/14:0, astaxanthin diester-20: 5/16:1, astaxanthin diester-18: 1/16:0, astaxanthin diester-16: 0/16:0, astaxanthin diester-22: 6/20:5, Astaxanthin diester-20: 5/18:1, astaxanthin monoester-18: 2, astaxanthin monoester-16: 0, astaxanthin monoester-18: 1, astaxanthin monoester-22: 6, astaxanthin monoester-20: 1, astaxanthin monoester-20: 5, astaxanthin monoester-16: 1, astaxanthin monoester-20: 4, astaxanthin diester-22: 6/16:0, adonixanthin diester-22: 6-20:6, and adonixanthin diester-22: 6-16: 0.
EXAMPLE 3 (method one)
A high-flux detection method for carotenoids in aquatic products comprises the following steps:
A. pretreatment:
(A1) weighing 1g of aquatic product to be detected, adding the aquatic product to be detected into 10mL of methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, ultrasonically extracting for 20min, and centrifuging at the rotating speed of 12000r/min at 5 ℃ for 8min to obtain an organic phase and residue;
(A2) taking the organic phase obtained in the step (A1), adding at least 4 times of volume of water into the organic phase to obtain a sample loading solution, purifying the sample loading solution by an HLB (hydrophile-lipophile balance) column to obtain a purified sample loading solution, concentrating the purified sample loading solution to less than 2.0ml by nitrogen blowing, and carrying out volume metering to 2.0ml by a methanol solution containing 0.1 wt% of HF to obtain a purified sample loading solution with a constant volume, wherein:
the HLB column purification adopts methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol as eluent for elution;
(A3) adding the residue obtained in the step (A1) into 10mL of methanol-dichloromethane solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, performing ultrasonic extraction for 20min, centrifuging for 7min at 6 ℃ at the rotating speed of 12000r/min, and obtaining an organic phase and residue, wherein:
in the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1: 1;
(A4) Taking the organic phase obtained in the step (A3), blowing nitrogen to concentrate until the volume is not changed, then fixing the volume to 2.0ml through a methanol solution containing 0.1 wt% of HF, adding 1g of neutral alumina powder into the organic phase, fully shaking, centrifuging for 8min at 6 ℃ at the rotating speed of 12000r/min, and obtaining supernatant and precipitate after completion;
(A5) mixing the purified sample solution with the constant volume obtained in the step (A2) and the supernatant obtained in the step (A4), and then filtering the mixture through a filter membrane with the pore diameter of 0.22 mu m to obtain a sample solution to be detected;
B. and (C) detecting the sample solution to be detected obtained in the step (A5) by using a high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrometer to obtain a high performance liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows:
high performance liquid phase parameters:
column ZORBAX Eclipse XDB C8 column 2.1mm x 100mm, particle size 3.5 μm;
mobile phase A: an aqueous solution containing 0.1% formic acid +2mM ammonium formate;
mobile phase B: a methanol solution containing 0.1 v/v% formic acid +15 v/v% isopropanol was injected at a flow rate of 0.8mL/min, a sample size of 5.0. mu.L, a split ratio of 1:4, and the gradient was as follows:
positive and negative ion mode gradient elution procedure
Mass spectrum parameters: the mass spectrum is subjected to full-scan measurement in a positive/negative ion conversion mode, and the mass range is as follows: m/z 200-1500, resolution 70000, and target value of automatic gain control 5 × e 5(ii) a Anion mode 2900V, positive ion mode 3800V, ion transfer tube temperature 350 ℃, desolvation gas nitrogen flow rate: 35L/h, flow rate of auxiliary gas nitrogen: 15L/h, the temperature of a gasification chamber is 400 ℃; respectively carrying out positive and negative ion correction on the instrument before the sample runs; the second stage adopts an automatic trigger mode, the resolution is 35,000, and the target value of automatic gain control is 2 × e5The collision energy range is 10-80%, and the retention time is +/-1.0 min according to the retention time of the target;
c data analysis
C1 plots standard curves for the standards:
preparing mixed standard working solutions with different concentrations by taking alpha-carotene, beta-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combretastatin, cryptoxanthin, diadinoxanthin, dihydrolutein, echinenone, fucoxanthin alcohol, neoxanthin, polymethacrylic acid alcohol, uric acid lactone, mussel flavin, zeaxanthin, scallop alcoholic ketone, diatoxanthine, hydroxyl echinenone, dinotefuran, pectic xanthin, cryptoxanthin, astaxanthin linoleate monoester and astaxanthin dipalmitate, testing according to the same conditions of the step B to obtain a high-efficiency liquid chromatogram of the standard working solution, and drawing a standard curve according to the concentration and peak area of each standard product to obtain a standard curve of the standard product;
C2 selecting standard curve of linoleic acid astaxanthin monoester or dipalmitoyl acid astaxanthin ester with similar structure according to the quantity of fatty acid chains for relative quantitative analysis of carotenoid mono-ester and carotenoid diester without standard substance;
c3, calculating the concentration of each component to be detected in the sample solution to be detected according to the high performance liquid chromatogram of the sample solution to be detected obtained in the step (B), the corresponding standard curve of the standard product, the linoleic acid astaxanthin monoester standard curve and the dipalmitate astaxanthin ester standard curve, and calculating the content of the carotenoid in the aquatic product to be detected according to a formula (1):
X＝C*V/m*1000
in the formula:
the content of the substance to be detected in the X-sample is mg/kg;
c-the concentration of the substance to be detected in the sample treatment solution is calculated according to a standard curve, a linoleic acid astaxanthin monoester standard curve or a dipalmitate astaxanthin ester standard curve, and the unit is mu g/L
V is volume of constant volume, unit is mL;
m-sample volume or mass in g.
Further, the aquatic product to be detected in the step (a1) is a shrimp.
Further, the concentrations of the different concentrations of the mixed standard working solution in step C1 were 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 200ng/mL, respectively.
Further, the carotenoid esters without standards in step C2 include astaxanthin diester-20: 3/20:1, astaxanthin diester-20: 3/18:2, astaxanthin diester-22: 6/18:1, astaxanthin diester-20: 4/20:5, astaxanthin diester-20: 5/20:5, astaxanthin diester-22: 6/16:0, astaxanthin diester-22: 6/16:1, astaxanthin diester-20: 5/16:0, astaxanthin diester-22: 6/14:0, astaxanthin diester-20: 5/16:1, astaxanthin diester-18: 1/16:0, astaxanthin diester-16: 0/16:0, astaxanthin diester-22: 6/20:5, Astaxanthin diester-20: 5/18:1, astaxanthin monoester-18: 2, astaxanthin monoester-16: 0, astaxanthin monoester-18: 1, astaxanthin monoester-22: 6, astaxanthin monoester-20: 1, astaxanthin monoester-20: 5, astaxanthin monoester-16: 1, astaxanthin monoester-20: 4, astaxanthin diester-22: 6/16:0, adonixanthin diester-22: 6-20:6, and adonixanthin diester-22: 6-16: 0.
The second method comprises the following steps:
A. the pretreatment steps are as follows:
(1) weighing 1.00g of sample, adding 10mL of methanol solution (containing 0.1% BHT), shaking for 5min, ultrasonically extracting for 20min, and centrifuging at high speed at low temperature of 12000r/min to obtain organic layer and residue; (2) taking out the supernatant, blowing nitrogen to concentrate to less than 2.0ml, and metering the volume of methanol (containing 0.1 wt% of HF) to 2.0 ml; (3) adding 10mL of methanol-dichloromethane (1:1, v: v, containing 0.1 wt% BHT) solution into the residue, shaking for 5min, ultrasonic extracting for 20min, and centrifuging at 12000r/min at high speed to obtain organic layer and residue; (4) collecting the upper organic phase, nitrogen-blowing to concentrate to near dry, diluting methanol (containing 0.1 wt% HF) to 2.0ml, and centrifuging at high speed at 12000r/min to obtain supernatant and precipitate; (5) the supernatants of (2) and (4) were combined and filtered through a 0.22 μm filter.
Step B and step C are the same as method one (embodiment 3), and are not described again.
The third method comprises the following steps:
A. the pretreatment steps are as follows:
(1) weighing 1.00g of sample, adding 10mL of acetone solution (containing 0.1 wt% of BHT), shaking for 5min, ultrasonically extracting for 20min, and centrifuging at a low temperature of 12000r/min at a high speed to obtain an organic layer and residue; (2) the extraction was repeated, the supernatants were combined, concentrated to <4.0ml by nitrogen blow, and the volume of methanol (containing 0.1% wtHF) was adjusted to 4.0ml, passed through a 0.22 μm filter.
Step B and step C are the same as method one (embodiment 3), and are not described again.
The method four comprises the following steps:
A. the pretreatment steps are as follows:
(1) weighing 1.00g of sample, adding 10mL of ethyl acetate (containing 0.1 wt% of BHT) solution, shaking for 5min, ultrasonically extracting for 20min, and centrifuging at low temperature of 12000r/min at high speed to obtain an organic layer and residue; (2) the extraction was repeated, the supernatants were combined, concentrated by nitrogen blow to near dryness, the volume of methanol (containing 0.1 wt% HF) was adjusted to 4.0ml, and the solution was filtered through a 0.22 μm filter.
Step B and step C are the same as method one (embodiment 3), and are not described again.
And (3) verification test:
by utilizing the established pretreatment method, the carotenoid species in the common aquatic product tiger shrimp is screened. The results show (see table below), where:
the method comprises the following steps: the method adopts fractional extraction (methanol solution and methanol-dichloromethane solution are sequentially extracted), purification is carried out step by step (the matrix property of extracting solution is subjected to purification treatment, HLB column purification and neutral alumina powder oil removal are combined), 50 carotenoids are detected in total, including 25 non-esterified carotenoids and 25 esterified carotenoids, and the total content value is as high as 2055 +/-199 mg/kg, so that the feasibility of the extraction and purification method is fully verified.
The second method comprises the following steps: due to lack of corresponding purification steps, the extract has strong matrix inhibition effect, so that low-content carotenoid can not be detected, 24 kinds of carotenoid including 10 kinds of non-esterified carotenoid and 14 kinds of esterified carotenoid can be detected together, and the total content value is 1052 +/-102 mg/kg.
The third method comprises the following steps: the pretreatment adopts medium-polarity acetone as an extracting solution, the extraction rate of low-polarity carotenoid ester cannot be ensured, 22 carotenoids are detected in total, including 16 non-esterified carotenoids and 6 esterified carotenoids, and the total content value is 716 +/-80.3 mg/kg; the same is true.
The method comprises the following steps: the pretreatment adopts low-polarity ethyl acetate as an extracting solution, and the synchronously extracted grease has strong ionization inhibiting effect, so that the content value of the carotenoid ester with low content is reduced or cannot be detected, meanwhile, the extraction rate cannot be ensured for the medium-polarity non-esterified carotenoid and metabolites thereof, 19 carotenoids are detected together, including 5 non-esterified carotenoids and 14 esterified carotenoids, and the total content value is 346 +/-29.6 mg/kg.
In conclusion, the method has strong compatibility with different aquatic product matrixes and compounds with different physicochemical properties, the carotenoid detected by the method is more abundant in variety and more accurate in content value, and the high flux of the detection method is fully verified by comparing the method with the traditional carotenoid detection method (method three and method four) (see the following table).
Carotenoid species and content detected under 4 treatment methods (n ═ 6)
"-" indicates a content of <0.05mg/kg
The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (3)
1. A high-flux detection method for carotenoids in aquatic products is characterized by comprising the following steps:
A. pretreatment:
(A1) weighing 1g of aquatic product to be detected, adding the aquatic product to be detected into 10mL of methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, ultrasonically extracting for 20min, and centrifuging for 5-10 min at the rotating speed of 12000r/min at the temperature of 4-10 ℃, thus obtaining an organic phase and residues;
(A2) taking the organic phase obtained in the step (A1), adding at least 4 times of volume of water into the organic phase to obtain a sample loading solution, purifying the sample loading solution by an HLB (hydrophile-lipophile balance) column to obtain a purified sample loading solution, concentrating the purified sample loading solution to less than 2.0ml by nitrogen blowing, and carrying out volume metering to 2.0ml by a methanol solution containing 0.1 wt% of HF to obtain a purified sample loading solution with a constant volume, wherein:
the HLB column purification adopts methanol solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol as eluent for elution;
(A3) Adding the residue obtained in the step (A1) into 10mL of methanol-dichloromethane solution containing 0.1 wt% of 2, 6-di-tert-butyl-p-cresol, oscillating for 5min, performing ultrasonic extraction for 20min, centrifuging for 5-10 min at 4-10 ℃ at the rotating speed of 12000r/min, and obtaining an organic phase and residue, wherein:
in the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1: 1;
(A4) taking the organic phase obtained in the step (A3), blowing nitrogen to concentrate until the volume is not changed, then fixing the volume to 2.0ml through a methanol solution containing 0.1 wt% of HF, adding 1g of neutral alumina powder, fully shaking, centrifuging at 4-10 ℃ for 5-10 min at a rotating speed of 12000r/min, and obtaining a supernatant and a precipitate after completion;
(A5) combining the purified sample solution obtained in the step (A2) after constant volume and the supernatant obtained in the step (A4), and then passing through a filter membrane with the aperture of 0.22 mu m to obtain a sample solution to be detected;
B. and (C) detecting the sample solution to be detected obtained in the step (A5) by using a high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrometer to obtain a high performance liquid chromatogram of the sample solution to be detected, wherein the instrument conditions are as follows:
high performance liquid phase parameters:
column ZORBAX Eclipse XDB C8 column 2.1mm x 100mm, particle size 3.5 μm;
Mobile phase A: an aqueous solution containing 0.1% formic acid +2mM ammonium formate;
mobile phase B: a methanol solution containing 0.1 v/v% formic acid +15 v/v% isopropanol was injected at a flow rate of 0.8mL/min, a sample size of 5.0. mu.L, a split ratio of 1:4, and the gradient was as follows:
positive and negative ion mode gradient elution procedure
Mass spectrum parameters: the mass spectrum is subjected to full-scan measurement in a positive/negative ion conversion mode, and the mass range is as follows: m/z 200-1500, resolution 70000, and target value of automatic gain control 5 × e5(ii) a The negative ion mode 2900V, the positive ion mode 3800V and the ion transmission tube temperature are 350 ℃, desolventizing gas nitrogen flow rate: 35L/h, flow rate of auxiliary gas nitrogen: 15L/h, the temperature of a gasification chamber is 400 ℃; respectively carrying out positive and negative ion correction on the instrument before the sample runs; the second stage adopts an automatic trigger mode, the resolution is 35,000, and the target value of automatic gain control is 2 × e5The collision energy range is 10-80%, and the retention time is +/-1.0 min according to the retention time of the target;
c data analysis
C1 plots standard curves for the standards:
preparing mixed standard working solutions with different concentrations by taking alpha-carotene, beta-carotene, astaxanthin, fucoxanthin, purple zeaxanthin, canthaxanthin, lutein, combretastatin, cryptoxanthin, diadinoxanthin, dihydrolutein, echinenone, fucoxanthin alcohol, neoxanthin, polymethacrylic acid alcohol, uric acid lactone, mussel flavin, zeaxanthin, scallop alcoholic ketone, diatoxanthine, hydroxyl echinenone, dinotefuran, pectic xanthin, cryptoxanthin, astaxanthin linoleate monoester and astaxanthin dipalmitate, testing according to the same conditions of the step B to obtain a high-efficiency liquid chromatogram of the standard working solution, and drawing a standard curve according to the concentration and peak area of each standard product to obtain a standard curve of the standard product;
C2 selecting standard curve of linoleic acid astaxanthin monoester or dipalmitoyl astaxanthin ester with similar structure according to the quantity of fatty acid chains for relative quantitative analysis of carotenoid mono-and diester without standard;
c3 calculating the concentration of each component to be detected in the sample solution to be detected according to the high performance liquid chromatogram of the sample solution to be detected obtained in the step (B), the corresponding standard curve of the standard product, the linoleic acid astaxanthin monoester standard curve and the dipalmitate astaxanthin ester standard curve,
calculating the carotenoid content in the aquatic product to be detected according to a formula (1):
X＝C*V/m*1000
in the formula:
the content of the substance to be detected in the X-sample is mg/kg;
the concentration of the substance to be measured in the C-sample treatment solution is calculated according to a standard curve, a linoleic acid astaxanthin monoester standard curve or a dipalmitate astaxanthin ester standard curve, and the unit is mug/L
V is volume of constant volume, and the unit is mL;
m-sample volume or mass in g, wherein:
the carotenoid esters without standards in step C2 include astaxanthin diester-20: 3/20:1, astaxanthin diester-20: 3/18:2, astaxanthin diester-22: 6/18:1, astaxanthin diester-20: 4/20:5, astaxanthin diester-20: 5/20:5, astaxanthin diester-22: 6/16:0, astaxanthin diester-22: 6/16:1, astaxanthin diester-20: 5/16:0, astaxanthin diester-22: 6/14:0, astaxanthin diester-20: 5/16:1, astaxanthin diester-18: 1/16:0, astaxanthin diester-16: 0/16:0, astaxanthin diester-22: 6/20:5, astaxanthin diester-20: 5/18:1, Astaxanthin monoester-18: 2, astaxanthin monoester-16: 0, astaxanthin monoester-18: 1, astaxanthin monoester-22: 6, astaxanthin monoester-20: 1, astaxanthin monoester-20: 5, astaxanthin monoester-16: 1, astaxanthin monoester-20: 4, astaxanthin diester-22: 6/16:0, adonixanthin diester-22: 6-20:6, and adonixanthin diester-22: 6-16: 0.
2. The method according to claim 1, wherein the aquatic product to be detected in step (A1) is one of fish, shrimp, crab and shell.
3. The method for high-throughput detection of carotenoids in aquatic products according to claim 1, wherein the concentrations of the mixed standard working solution of different concentrations in step C1 are 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 200ng/mL, respectively.
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